Thesis (Ph.D.) - University of Glasgow, 2008. / Ph.D. thesis submitted to the Faculty of Medicine, Division of Cancer Sciences and Molecular Pathology, University of Glasgow, 2007. Includes bibliographical references. Print version also available.

In eukaryotes, the SET1 family of methyltransferases carry out the methylation of Lysine 4 on Histone H3. Alone, these enzymes exhibit low enzymatic activity and require the presence of additional regulatory proteins, which include RbBP5, Ash2L, WDR5 and DPY-30, to stimulate their catalytic activity. While previous structural studies established the structural basis underlying the interaction between RbBP5, Ash2L and WDR5, the formation of the Ash2L/DPY-30 complex remains elusive. Here we report the crystal structure of the Ash2L/DPY-30 complex solved at 2.2Å. Our results show that a Cterminal amphipathic α-helix on Ash2L makes several hydrophobic interactions with the DPY-30 homodimer. Moreover, the structure reveals that a tryptophan residue on Ash2L, which directly precedes its C-terminal amphipathic α-helix, makes key interactions with one of DPY-30 α-helix. Finally, biochemical studies of Ash2L revealed a hitherto unknown ability of this protein to bind anionic lipids.

Thesis advisor: Thomas N. Seyfried / Two major metabolic phenomena observed in cancer cells include the Warburg effect and Crabtree effect. The Crabtree effect is the in vitro inhibition of respiration by glucose. The influence of glucose on the oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) of tumorigenic RAW264.7 and VM-M3 macrophage cells, as well as non-tumorigenic BV-2 microglia cells, was studied using the Seahorse XF96 extracellular flux analyzer. RAW264.7, VM-M3, and BV-2 cells incubated in glucose medium displayed a significantly lower OCR and higher ECAR compared to cells incubated in no glucose medium. Furthermore, when glucose medium was added to the RAW264.7 and BV-2 cells in real-time using the Seahorse XF96 injection ports, a rapid decrease in OCR and increase and ECAR was observed. Therefore, RAW264.7, VM-M3, and BV-2 cells display a robust Crabtree effect in vitro, as assessed by OCR and ECAR. Additionally, it is important to consider the Crabtree effect when studying in vitro energy metabolism of all cell and tissue types. It was also found that the elimination of the Crabtree effect through glucose deprivation resulted in dynamic cardiolipin (CL) fatty acid changes in VM-M3 cells. VM-M3 cells incubated in 10 mM glucose medium for four hours displayed a short-chain, saturated (immature) CL fatty acid composition, while VM-M3 cells incubated in no glucose media for four hours displayed long-chain, unsaturated (mature) CL fatty acid composition. Cardiolipin (CL) is a phospholipid highly enriched in the inner mitochondrial membrane. Mature, long-chain, unsaturated CL molecular species are involved in maintaining mitochondrial function and membrane integrity. Overall, these data suggest that CL fatty acid composition may function as a structural component of the Crabtree effect in vitro. The Warburg effect, or aerobic glycolysis, is the observation that tumor cells consume less oxygen and more glucose than normal, untransformed cells in the presence of oxygen. It has been shown that immune cells display a Warburg effect upon activation by changing their core metabolism from oxidative phosphorylation to glycolysis. In this study, it was observed that both RAW264.7 macrophage cells and BV-2 microglia cells display a significantly lower OCR and higher ECAR following LPS-activation. However, this observation is dependent on the concentration of LPS. Therefore, these data suggest that both RAW264.7 and BV-2 cells display a LPS concentration-dependent change in metabolism from oxidative phosphorylation to glycolysis upon LPS-activation in vitro. The in vitro lipid profiles that resulted from the Crabtree effect and the LPS-activated Warburg effect were also studied in the RAW264.7 cell line. The lipids phosphatidylserine (PS) and cardiolipin (CL) displayed the most robust changes in the RAW264.7 cells. Both PS and CL have been shown to be associated with cellular respiration. / Thesis (MS) — Boston College, 2016. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Biology.

The blood-brain barrier (BBB) formed by the brain capillary endothelial cells provides a protective barrier between the systemic blood and the extracellular environment of the central nervous system. Since most fatty acids in the brain enter from the blood, we examined the mechanism of permeability of various fatty acids across primary human brain microvessel endothelial cells (HBMEC). Cardiolipin (CL), a major mitochondrial phospholipid involved in energy metabolism in mammalian mitochondria, and fatty acid transport protein-1 (FATP-1) may regulate the intracellular level of fatty acyl-Coenzyme A’s. Since fatty acids are required for oxidative phosphorylation via mitochondrial oxidation, we also examined the effect of altering FATP-1 levels on CL biosynthesis. The permeability of radiolabeled fatty acids was determined using confluent cells grown on Transwell® inserts following inhibition of various fatty acid transporters. The passage of [1-14C]oleate across confluent HBMEC monolayers was significantly enhanced when fatty acid free albumin was present in the basolateral media. Knockdown of FATP-1, FATP-4, fatty acid translocase/CD36, or fatty acid binding protein 5 significantly decreased permeability of a number of radiolabeled fatty acids across the HBMEC monolayer from either apical as well as basolateral sides. The findings indicate that transport of some fatty acids across HBMEC is, in part, a transcellular process mediated by fatty acid transport proteins. Next, HEK 293 cells were used as a model to determine the effect of altering FATP-1 levels on CL. HEK-293 mock- and FATP-1 siRNA-transfected cells or mock and FATP-1 expressing cells were incubated for 24 h with 0.1 mM oleate bound to albumin (1:1 molar ratio) then incubated for 24 h with 0.1 mM [1,3-3H]glycerol and radioactivity incorporated into CL determined. FATP-1 siRNA-transfected cells exhibited reduced FATP-1 mRNA and increased incorporation of [1,3-3H]glycerol into CL (2-fold, p<0.05) compared to controls indicating elevation in de novo CL biosynthesis. In contrast, expression of FATP-1 resulted a reduction in incorporation of [1,3-3H]glycerol into CL (65%, p<0.05) indicating reduced CL synthesis. In addition, in vitro cytidine-5’-diphosphate-1,2-diacyl-sn-glycerol synthetase (CDS) activity was reduced by exogenous addition of oleoyl-Coenzyme A. The data indicate that CL de novo biosynthesis may be regulated by FATP-1 through CDS-2 expression in HEK 293 cells.

The human genome project has allowed for the rapid identification of a large number of protein families based on similarities in their genetic sequences. In the present study, I report the functional characterization of a 44 kDa protein that functions in cardiolipin synthesis and remodeling. Although it is present in most tissues, it is abundant in multiple brain regions including olfactory bulbs, hippocampus, cerebellum, cortex, and brain stem, and is detectable in both primary neurons and glial cells. In assays performed in vitro, this protein significantly increased the incorporation of [14C]oleoyl-CoA into phosphatidylinositol and CL using either lysophosphatidylinositol, or monolysocardiolipin or dilysocardiolipin as acyl acceptors, respectively. This protein did not display significant acyltransferase activity with a number of other lysophospholipid acyl acceptors. Overexpressing this enzyme in HEK-293 cells increased total CL content, but did not significantly affect levels of other glycerophospholipids. Analysis of the fatty acyl profile of CL from cells overexpressing this protein indicated increased total saturated fatty acids, particularly stearate, palmitate, and myristate, and increased levels of n-3 polyunsaturated fatty acids α-linoleic acid (18:3n-3), eicosatrienoic acid (20:3n-3), and eicosapentanoic acid (20:5n-3). In accordance with its observed role in CL remodeling, subcellular localization of this protein was predominately mitochondrial. This protein is also regulated during embryogenesis, and in varying metabolic states.

The blood-brain barrier (BBB) formed by the brain capillary endothelial cells provides a protective barrier between the systemic blood and the extracellular environment of the central nervous system. Since most fatty acids in the brain enter from the blood, we examined the mechanism of permeability of various fatty acids across primary human brain microvessel endothelial cells (HBMEC). Cardiolipin (CL), a major mitochondrial phospholipid involved in energy metabolism in mammalian mitochondria, and fatty acid transport protein-1 (FATP-1) may regulate the intracellular level of fatty acyl-Coenzyme A’s. Since fatty acids are required for oxidative phosphorylation via mitochondrial oxidation, we also examined the effect of altering FATP-1 levels on CL biosynthesis. The permeability of radiolabeled fatty acids was determined using confluent cells grown on Transwell® inserts following inhibition of various fatty acid transporters. The passage of [1-14C]oleate across confluent HBMEC monolayers was significantly enhanced when fatty acid free albumin was present in the basolateral media. Knockdown of FATP-1, FATP-4, fatty acid translocase/CD36, or fatty acid binding protein 5 significantly decreased permeability of a number of radiolabeled fatty acids across the HBMEC monolayer from either apical as well as basolateral sides. The findings indicate that transport of some fatty acids across HBMEC is, in part, a transcellular process mediated by fatty acid transport proteins. Next, HEK 293 cells were used as a model to determine the effect of altering FATP-1 levels on CL. HEK-293 mock- and FATP-1 siRNA-transfected cells or mock and FATP-1 expressing cells were incubated for 24 h with 0.1 mM oleate bound to albumin (1:1 molar ratio) then incubated for 24 h with 0.1 mM [1,3-3H]glycerol and radioactivity incorporated into CL determined. FATP-1 siRNA-transfected cells exhibited reduced FATP-1 mRNA and increased incorporation of [1,3-3H]glycerol into CL (2-fold, p<0.05) compared to controls indicating elevation in de novo CL biosynthesis. In contrast, expression of FATP-1 resulted a reduction in incorporation of [1,3-3H]glycerol into CL (65%, p<0.05) indicating reduced CL synthesis. In addition, in vitro cytidine-5’-diphosphate-1,2-diacyl-sn-glycerol synthetase (CDS) activity was reduced by exogenous addition of oleoyl-Coenzyme A. The data indicate that CL de novo biosynthesis may be regulated by FATP-1 through CDS-2 expression in HEK 293 cells.

The blood brain barrier (BBB), formed by endothelial cells lining the lumen of the brain capillaries, is a restrictively permeable interface that only allows transport of specific compounds into the brain. Cardiolipin (CL) is a mitochondrial- specific phospholipid known to be required for the activity and integrity of the respiratory chain. The current study examined the role of cardiolipin in maintaining an optimal mitochondrial function that may be necessary to support the barrier properties of the brain microvessel endothelial cells (BMECs). Endothelial cells have been suggested to obtain most of their energy through an-aerobic glycolysis based on studies of cells that were obtained from the peripheral vasculatures. However, here, we showed that the adult human brain capillary endothelial cell line (hCMEC/D3) appeared to produce ~60% of their basal ATP requirement through mitochondrial oxidative phosphorylation. In addition, RNAi mediated knockdown of the CL biosynthetic enzyme cardiolipin synthase (CLS), although did not grossly affect the mitochondrial coupling efficiency of the hCMEC/D3 cells, did seem to reduce their ability to increase their mitochondrial function under conditions of increased demand. Furthermore, the knockdown appeared to have acted as a metabolic switch causing the hCMEC/D3 cells to become more dependent on glycolysis. These cells also showed increase in [3H]-2-deoxyglucose uptake under a low glucose availability condition, which might have served as a mechanism to compensate for their reduced energy production efficiency. Interestingly, the increase in glucose uptake appeared correlated to an increase in [3H]-2-deoxyglucose glucose transport across the knockdown confluent hCMEC/D3 monolayers grown on Transwell® plates, which was used in our study as an in vitro model for the human BBB. This suggests that changes in the brain endothelial energy status may play a role in regulating glucose transport across the BBB. These observations, perhaps, also explain why the brain capillary endothelial cells were previously observed to possess higher mitochondrial content than those coming from non-BBB regions (Oldendorf et al. 1977).

Atherosclerosis is an insidious disease with serious morbidity and mortality including ischemic heart disease, stroke, and myocardial infarction. This condition is progressive and can start early in life eventually leading to large plaques and arterial occlusion. Two key components of this process are the immune system and lipids; in particular, LDL which accumulates within the arterial walls and macrophages which recognize and engulf oxidized-LDL (oxLDL) to form foam cells. Knowing that certain antibodies directed against bacterial antigens such as phosphorylcholine (PC) and cardiolipin (CL) show opsonizing cross-reactivity with oxLDL it can be proposed that there is a link between immune responses to periodontal bacteria and atherosclerosis. The aim of this investigation was to determine whether periodontal bacteria are capable of inducing serum antibodies potentially involved in cardiovascular diseases; specifically, IgG anti-PC, IgG anti-CL, and IgM anti-CL. To test this, 17 subjects with chronic periodontitis received scaling and root planing in conjunction with blood sample analysis to determine if periodontal instrumentation resulted in changes in these serum antibodies. If plaque bacteria are responsible for an immune response then serum levels of these antibodies should decrease following periodontal therapy. We found that serum levels of IgG anti-PC, IgG anti-CL, and IgM anti-CL decreased following periodontal scaling and root planing but the change was significant only for IgG anti-PC (P 0.045). Serum levels of IgM anti-CL approached significance (P 0.054). The results support the hypothesis that the immune response to periodontal bacterial microflora contributes to serum concentrations of antiphospholipid antibodies.

Thesis advisor: Thomas Seyfried / Lipids, in all their forms from structural components of the membranes (phosphoglycerides, glycolglycerolipids) to signaling molecules (IP3, DAG, prostaglandins, etc.,) post-translational modification of proteins (palmitoylated, farnesylated, prenylated, and GPI anchoring) play an essential role in cancer cell survival, proliferation, and metastasis. Alteration in structural lipids can impair transport, and signaling cascades. Abnormalities in lipids, such as cardiolipin (Ptd2Gro), impair mitochondrial function, bioenergetics, and could play a role in precipitatting the high incidence of spontaneous tumors in VM/Dk mice. This thesis explores the role of glucose and glutamine in their incorporation into lipids in the VM-M3 murine glioblastoma cell line as well as the inheritance of brain cardiolipin fatty acids abnormalities in VM/Dk mice. I used labeled [14C]-U-D-glucose and [14C]-U-L-glutamine to examine the profile of de novo lipid biosynthesis in the VM-M3 cell line. The major lipids synthesized included phosphatidylcholine (PtdCho), phosphatidylethanolamine (EtnGpl), phosphatidylinositol (PtdIns), phosphatidylserine (PtdSer), sphingomyelin (CerPCho), bis(monoacylglycero)phosphate (BMP) / phosphatidic acid (PtdOH), cholesterol (C), Ptd2Gro, and the gangliosides. The data show that the incorporation of labeled glucose and glutamine into synthesized lipids was dependent on the type of growth environment, and that the VM-M3 glioblastoma cells could acquire lipids, especially cholesterol, from the external environment for growth and proliferation. In addition, this thesis also explores and evaluates the abnormality of Ptd2Gro fatty acid composition in VM mice in comparison to B6 mice. Although previously reported, I confirmed the finding in the abnormal cardiolipin fatty acid composition in the VM mice. The abnormal brain cardiolipin fatty acid composition was found to be inherited as an autosomal dominant trait in reciprocal B6 x VM F1 hybrids for both male and female. Impaired cognitive awareness under hypoxia observed for the VM mice and reciprocal F1 hybrids is associated with abnormalities in neural lipid composition. / Thesis (PhD) — Boston College, 2014. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Biology.

A interação do citocromo c com a cardiolipina ocorre por interações eletrostáticas e hidrofóbicas. A formação do complexo citocromo c/ cardiolipina promove uma pequena mudança estrutural na proteína, que proporciona atividade peroxidásica ao citocromo c e consequentemente capacidade de oxidar substratos orgânicos, incluindo a cardiolipina. A oxidação da cardiolipina acompanhada da inserção de um grupo peróxido vem sendo relacionada à perda da interação hidrofóbica entre o complexo citocromo c/cardiolipina, que resulta no desligamento do citocromo c da membrana e na sua saída do espaço intermembranas para o citosol, onde essa proteína induz a cascata de apoptose. Neste trabalho foi avaliada a ligação do citocromo c a lipossomos contendo cardiolipina oxidada e a reatividade desta proteína com o mono-hidroperóxido da cardiolipina (TLCL(OOH)1) presente na membrana. Nossos dados mostraram que ocorre uma diminuição significativa na ligação do citocromo c a membrana oxidadas apenas quando 100% da cardiolipina presente na membrana está na forma de TLCL(OOH)1, condição que extrapolaria o que seria esperado para o sistema biológico. Análises por SDS-PAGE revelaram que o citocromo c sofre agregação na presença de membranas contendo TLCL(OOH)1, indicando que a proteína reage com este peróxido. De fato, determinamos a velocidade de reação do citocromo c com o TLCL(OOH)1 e com hidroperóxido do ácido linoléico, inseridos em membrana contendo cardiolipina (9,58 ± 0,16 x 102 M-1.s-1 e 6,91 ± 0,30 x 102 M-1.s-1, respectivamente). As velocidades de reação com os peróxidos de lipídio foram pelo menos 10 vezes superiores à velocidade medida com o peróxido de hidrogênio (5,91 ± 0,18 x101 M-1.s-1). Assim, mostramos que o citocromo c liga-se à membrana contendo hidroperóxido de cardiolipina e que reage com o mesmo promovendo a formação de agregado protéico de alto peso molecular / The interaction of cytochrome c with cardiolipin is promoted by electrostatic and hydrophobic interactions. The cytochrome c / cardiolipin complex formation causes structural changes in the protein that activates cytochrome c peroxidase activity, giving it the ability to oxidize organic substrates, including cardiolipin. The oxidation of cardiolipin coupled with a peroxide group insertion has been related to the loss of hydrophobic interactions between the cytochrome c / cardiolipin complex, resulting in cytochrome c release from the membrane and in its translocation from intermembranes space to cytosol, where this protein induces apoptosis cascade. In this work the binding of cytochrome c to liposomes containing oxidized cardiolipin and its reactivity with the membrane mono-hydroperoxides (TLCL(OOH)1) were evaluated. Our data showed a significant decrease in cytochrome c binding to oxidized membranes only when 100% of the membrane cardiolipin is in the TLCL(OOH)1 form, a condition that would extrapolate the expected concentrations that would be found in a biological system. SDS-PAGE analysis revealed that cytochrome c undergoes aggregation in the presence of membranes containing TLCL(OOH)1, indicating that this protein reacts with the peroxide. In fact, we determined the rate of cytochrome c reaction with TLCL(OOH)1 and linoleic acid hydroperoxide inserted into cardiolipin containing membranes (9.58 ± 0.16 x 102 M-1s-1 and 6.91 ± 0.30 x 102 M-1s-1,respectively). The reaction rates obtained with lipid peroxides were at least 10 times higher than that obtained with hydrogen peroxide (5.91 ± 0.18 x 101 M-1s-1).Thus we show that cytochrome c binds to membrane containing cardiolipin hydroperoxides and reacts with it promoting the formation of high molecular weight protein aggregates.